In response to phagocytic stimuli, human neutrophils undergo a massive respiratory burst in which oxygen is converted to superoxide, hydrogen peroxide, and hydroxyl radical. While these agents usually serve to kill ingested bacteria, their inadvertent production by neutrophils has been implicated the pathogenesis of certain diseases such as adult respiratory distress syndrome. It is, therefore, not surprising that the activation of this respiratory burst is tightly regulated. The studies proposed in this grant are part of a long range goal to eludicate the biochemical mechanisms which regulate superoxide production in human neutrophils. At the center of this mechanism is NADPH oxidase, the enzyme which generates superoxide and which is converted from an inactive to an active form when the neutrophil is stimulated. Our preliminary studies indicate that there is an activation pathway which ultimately triggers the oxidase. Only limited details are known regarding this pathway, however in this grant we propose to study the following biochemical changes as they pertain to activation: 1) the role of intracellular second messengers, in particular calcium, free fatty acids, and arachidonic acid metabolites; 2) the role of covalent modification of the resting oxidase by either limited proteolysis or phosphorylation following stimulation of the cells; 3) the possibility that the oxidase is composed of two or more subunits, each catalytically inactive which are assembled upon stimulation to form an active enzyme complex. We will study these processes in several systems. First, we have already begun to study activation on intact neutrophils by fatty acids and will extend these studies. Second, we have recently developed a technique for permeabilizing resting neutrophils that will permit extensive experimental manipulation of the intracellular milieu. We found that these cells retain their ability to be activated by conventional stimuli. Third, we will attempt to activate the oxidase in homogenates of resting neutrophils, a technique yet to be developed. Finally, we will attempt to raise monoclonal antibodies against NADPH oxidase using a screening assay which selects, for antibodies that inhibit enzyme actvity. If successful, we will use the antibody to isolate the oxidase complex and study its components in neutrophils from normal individuals and patients with chronic granulomatous disease. These studies may enable us to construct an activation pathway which might ultimately permit the development of anti-inflammatory agents capable of selectively interupting various steps in the pathway.